Eisenia fetida Protease-III-1 Functions in Both Fibrinolysis and Fibrogenesis

The fibrinolytic function of earthworm protease-III-1 (Ef P-III-1) has been studied in recent years. Here, we found that Ef P-III-1 acted not only in fibrinogenolysis, but also in fibrogenesis. We have used Ef P-III-1 to hydrolyze fibrinogen, and to activate plasminogen and prothrombin. Based on the N-terminal sequences of the hydrolytic fragments, Ef P-III-1 was showed to specifically recognize the carboxylic sites of arginine and lysine. Analyses by fibrinogenolysis mapping and amino acid sequencing revealed that the isozyme could cleave the alpha, beta, and gamma chains of fibrinogen, showing a high α-fibrinogenase, moderate β-fibrinogenase, and low γ-fibrinogenase activities. Interestingly, Ef P-III-1 activated plasminogen and released active plasmin, suggesting a tPA-like function. Furthermore, Ef P-III-1 showed a factor Xa-like function on prothrombin, producing alpha-thrombin. The function in both activating prothrombin and catalyzing fibrinogenolysis suggests that Ef P-III-1 may play a role in the balance between procoagulation and anticoagulation

An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells

<p>Abstract</p> <p>Background</p> <p>Virus-binding activity is one of the important functions of fibronectin (FN). It has been reported that a high concentration of FN in blood improves the transmission frequency of hepatitis viruses. Therefore, to investigate a protease that hydrolyzes FN rapidly is useful to decrease the FN concentration in blood and HBV infection. So far, however, no specific protease digesting FN in serum has been reported.</p> <p>Methods</p> <p>We employed a purified earthworm protease to digest serum proteins. The rapidly cleaved protein (FN) was identified by MALDI-TOF MS and western blotting. The cleavage sites were determined by N-terminus amino acid residues sequencing. The protease was orally administrated to rats to investigate whether serum FN <it>in vivo </it>became decreased. The serum FN was determined by western blotting and ELISA. In cytological studies, the protease was added to the medium in the culture of HepG2.2.15 cells and then HBsAg and HBeAg were determined by ELISA.</p> <p>Results</p> <p>The protease purified from earthworm <it>Eisenia fetida </it>was found to function as a fibronectinase (FNase). The cleavage sites on FN by the FNase were at R and K, exhibiting a trypsin alkaline serine-like function. The earthworm fibronectinase (EFNase) cleaved FN at four sites, R₂₅₉, R₁₀₀₅, K₁₅₅₇and R₂₀₃₉, among which the digested fragments at R₂₅₉, K₁₅₅₇and R₂₀₃₉were related to the virus-binding activity as reported. The serum FN was significantly decreased when the earthworm fibronectinase was orally administrated to rats. The ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.</p> <p>Conclusion</p> <p>The earthworm fibronectinase (EFNase) cleaves FN much faster than the other proteins in serum, showing a potential to inhibit HBV infection through its suppressing the level of HBeAg. This suggests that EFNase is probably used as one of the candidates for the therapeutic agents to treat hepatitis virus infection.</p

Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells

<p>Abstract</p> <p>Background</p> <p>D-ribose in cells and human serum participates in glycation of proteins resulting in advanced glycation end products (AGEs) that affect cell metabolism and induce cell death. However, the mechanism by which D-ribose-glycated proteins induce cell death is still unclear.</p> <p>Results</p> <p>Here, we incubated D-ribose with bovine serum albumin (BSA) and observed changes in the intensity of fluorescence at 410 nm and 425 nm to monitor the formation of D-ribose-glycated BSA. Comparing glycation of BSA with xylose (a control for furanose), glucose and fructose (controls for pyranose), the rate of glycation with D-ribose was the most rapid. Protein intrinsic fluorescence (335 nm), Nitroblue tetrazolium (NBT) assays and Western blotting with anti-AGEs showed that glycation of BSA incubated with D-ribose occurred faster than for the other reducing sugars. Protein intrinsic fluorescence showed marked conformational changes when BSA was incubated with D-ribose. Importantly, observations with atomic force microscopy showed that D-ribose-glycated BSA appeared in globular polymers. Furthermore, a fluorescent assay with Thioflavin T (ThT) showed a remarkable increase in fluorescence at 485 nm in the presence of D-ribose-glycated BSA. However, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose. This suggests that glycation with D-ribose induced BSA to aggregate into globular amyloid-like deposits. As observed by Hoechst 33258 staining, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) activity assay, flow cytometry using Annexin V and Propidium Iodide staining and reactive oxygen species (ROS) measurements, the amyloid-like aggregation of glycated BSA induced apoptosis in the neurotypic cell line SH-SY5Y.</p> <p>Conclusion</p> <p>Glycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells.</p

Three-Dimensional Culture of Hybridoma Cells Secreting Anti-Human Chorionic Gonadotropin by a New Rolling Culture System

Cell growth rate and production of monoclonal antibody (MAb) of hybridoma cells producing anti-human chorionic gonadotropin (hCG) MAb have been used as investigation criteria in double-mouthed rolling bottle (DMRB). Compared with T-flask cell culture, both of the cell number and MAb production increased by approximately 42.5% when the medium was supplemented with 5% fetal calf serum (FCS) and DMRB rotated at 2 turns per minute. Yield of MAb was experimentally related to the number of viable cells. Interestingly, MAb yield was four times as high as that cultured in T-flask in the first 24 hours, and about 75% yield of total MAb was secreted by 48 hours during the culture. It appears that the promoted cell growth and MAb yield are resulted from the three-dimensional growth of hybridoma cells under a suitably revolving condition

Amyloid-like aggregates of neuronal tau induced by formaldehyde promote apoptosis of neuronal cells

BACKGROUND: The microtubule associated protein tau is the principle component of neurofibrillar tangles, which are a characteristic marker in the pathology of Alzheimer's disease; similar lesions are also observed after chronic alcohol abuse. Formaldehyde is a common environmental contaminant and also a metabolite of methanol. Although many studies have been done on methanol and formaldehyde intoxication, none of these address the contribution of protein misfolding to the pathological mechanism, in particular the effect of formaldehyde on protein conformation and polymerization. RESULTS: We found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of ~0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron microscopy and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. The amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau. CONCLUSION: The results suggest that low concentrations of formaldehyde can induce human tau protein to form neurotoxic aggregates, which could play a role in the induction of tauopathies

A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the Xenopus retina

<p>Abstract</p> <p>Background</p> <p><it>Otx </it>genes, orthologues of the <it>Drosophila orthodenticle </it>gene (<it>otd</it>), play crucial roles in vertebrate brain development. In the <it>Xenopus </it>eye, <it>Xotx2 </it>and <it>Xotx5b </it>promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by <it>Drosophila </it>OTD, we performed an <it>in vivo </it>molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of <it>Xotx2</it>, <it>Xotx5b </it>and <it>otd</it>.</p> <p>Results</p> <p>We identified a small 8–10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and <it>vice versa</it>. Moreover, the insertion of this box into <it>Drosophila </it>OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box.</p> <p>Conclusion</p> <p>Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of <it>Drosophila </it>into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. We propose that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis.</p

Influencing of serum inflammatory factors on IVF/ICSI outcomes among PCOS patients with different BMI

IntroductionOverweight and obese are important factors leading to the occurrence of long-term complications in women with polycystic ovary syndrome (PCOS). There has been controversy over whether dissatisfaction with pregnancy outcomes in PCOS patients is influenced by chronic inflammatory status or obesity. This retrospective study analyzed the levels of inflammatory factors in PCOS patients with different body mass index (BMI) groups and effective predictors of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) pregnancy outcomes.MethodsThere were 273 women with PCOS diagnosed who completed serum inflammatory factors test between January 2017 and June 2022 were selected. The data of 7,649 infertility PCOS patients who received their first IVF/ICSI treatment in the Reproductive Center of Peking University Third Hospital during the period of the study were collected. Finally, 92 PCOS patients were included in the high BMI group, while 97 patients were included in the normal BMI group. Baseline characteristics were collected and the pregnancy outcomes were compared among the two groups. Then, serum inflammatory factors’ effect on IVF/ICSI pregnancy outcomes were analyzed with age, anti-Mullerian Hormone (AMH) and BMI adjusted.ResultsPCOS patients in the high BMI group significantly had a lower number of oocytes retrieved and good quality embryos. The high BMI group PCOS patients had higher levels of IL-6 and lower cumulative clinical pregnancy and live birth rates. The level of GM-CSF was higher in the first cycle transfer and cumulative miscarriage group. High TNF-α was negatively correlated with the first transfer cycle and cumulative clinical pregnancy rates after age, AMH and high BMI adjusted. In addition, the cumulative live birth rate was negatively correlated with high IL-6, but the first cycle transfer and cumulative live birth rates were positively correlated with high IL-1β.DiscussionFor PCOS patients, in addition to BMI, attention should also be paid to inflammatory indicators. High levels of TNF-α and IL-6 were negatively correlated with pregnancy outcomes, but high IL-1β was positively correlated with live birth rates among PCOS patients. The level of GM-CSF was higher in miscarriage PCOS patients

New Equivalent Linear Impact Model for Simulation of Seismic Isolated Structure Pounding against Moat Wall

Base-isolated buildings subjected to extreme earthquakes or near-fault pulse-like earthquakes can exceed their design gap distance and impact against the surrounding moat wall. Based on equating energy dissipation and maximum collision compression deformation of isolated structure with the Hertz-damp model and Kevin-Voigt model in the process of collision, an equivalent linear impact model (ELIM) is proposed to better predict impact response of seismic isolated structure. The formula of the equivalent linear stiffness of ELIM is theoretically derived. The effectiveness of ELIM is verified by comparing the results of numerical analyses with the results of pounding experiments. Four near-fault earthquakes are selected to validate rationality and accuracy of the proposed model using numerical analysis. The results indicate that the proposed linear model can nearly capture impact behavior of isolated structure in simulating the pounding-involved structural response

New Equivalent Linear Impact Model for Simulation of Seismic Isolated Structure Pounding against Moat Wall

Base-isolated buildings subjected to extreme earthquakes or near-fault pulse-like earthquakes can exceed their design gap distance and impact against the surrounding moat wall. Based on equating energy dissipation and maximum collision compression deformation of isolated structure with the Hertz-damp model and Kevin-Voigt model in the process of collision, an equivalent linear impact model (ELIM) is proposed to better predict impact response of seismic isolated structure. The formula of the equivalent linear stiffness of ELIM is theoretically derived. The effectiveness of ELIM is verified by comparing the results of numerical analyses with the results of pounding experiments. Four near-fault earthquakes are selected to validate rationality and accuracy of the proposed model using numerical analysis. The results indicate that the proposed linear model can nearly capture impact behavior of isolated structure in simulating the pounding-involved structural response